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PubMed1985

Structural and immunological relationships among mammalian arylsulfatase A enzymes.

Structural and immunological properties of numerous arylsulfatase A enzymes (EC 3.1.6) were examined in order to assess the relationships among these enzymes in animals. Arylsulfatase A enzymes from all animals bind to a Concanavalin A-Sepharose column, consistent with the conclusion that they are all glycoproteins. At pH 7.5 the apparent mol. wts of the enzymes are 80-182 kDa, while at pH 4.5 the mammalian arylsulfatase A enzymes dimerize and exhibit apparent mol. wts in the range of 297-348 kDa, but the enzymes from opossum and other lower classes of animals do not aggregate at pH 4.5. The mammalian arylsulfatase A enzymes, which aggregate at pH 4.5, also bind to rabbit liver arylsulfatase A monomers immobilized on an Affi-Gel 10 matrix. The arylsulfatase A enzymes that were studied all exhibit the anomalous kinetic behavior regarded as characteristic of these enzymes. However, not all of the inactivated enzymes are reactivated by sulfate ions. Goat antiserum raised against homogeneous rabbit liver arylsulfatase A cross-reacts with all of the mammalian enzymes in Ouchterlony gel diffusion experiments, whereas the enzymes from lower classes of animals do not cross-react. Quantitative immunoprecipitation experiments demonstrate that the mammalian enzymes are very similar to each other, with greater than 60% primary sequence homology indicated, while arylsulfatase A from opossum and other lower classes of animals show only a partial immunological similarity with the mammalian enzymes. Taken together, the data suggest that the active site of the enzyme and the structural features of the protein are highly conserved during the evolution of the enzyme molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Acylating agents as enzyme inhibitors and understanding their reactivity for drug design.

A series of bicyclic trans-fused gamma-lactones and gamma-lactams have been previously described for the inhibition of human neutrophil elastase and as possible development candidates. During the discovery program, it had been assumed that their acylating power was due in part to the inherent strain energy in the bicyclic structure that was released upon ring opening. This is now shown not to be the case, and in fact, these compounds are no more reactive than simple but analogous gamma-lactams and gamma-lactones. The strain energy is not released in the transition state for alkaline hydrolysis or alcoholysis because the reaction proceeds with rate-limiting formation of the tetrahedral intermediate. A reactivity index of k(OH) is proposed as a simple guide to determine the usefulness of a potential inhibitor as an enzyme acylating agent.

Acylation↗
PubMed1997

Increasing trend of HIV seroprevalence among pulmonary tuberculosis patients in Pune, India.

A total of 4618 tuberculosis patients attending the TB clinic at the Sassoon General Hospitals, Pune between 1991 and 1996 were screened for anti-HIV antibodies. Of these 694 were found reactive in enzyme immuno assay (EIA) and 624 were further confirmed by a second test, either rapid EIA or Western blot. HIV-1 reactivity was predominant among tuberculosis patients with HIV-2 reactivity appearing only in 1995. HIV-2 seroreactivity accounted for 0.54 and 1.02 per cent of all HIV reactive samples in 1995 and 1996. HIV-1 and HIV-2 dual reactivity accounted for 1.63 and 2.04 per cent of all infections in 1995 and 1996. The overall seroprevalence of HIV among newly diagnosed tuberculosis patients rose from 3.2 per cent in 1991 to 20.1 per cent in 1996.

HIV Seroprevalence↗
PubMed1985

Biochemical and biological characteristics of epitopes on the E1 glycoprotein of western equine encephalitis virus.

Antigenic determinants identified by monoclonal antibodies (Mabs) on the E1 glycoprotein of western equine encephalitis (WEE) virus have been characterized by their serological activity, requirements for secondary structure, expression on the mature virion, and their role in protecting animals from WEE virus challenge. On the basis of a cross-reactivity enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition assay, eight antigenic determinants (epitopes) on the E1 glycoprotein have been identified, ranging in reactivity from WEE-specific to alphavirus group reactive. No neutralization of virus infectivity was demonstrable with any of the Mabs. An alphavirus group-reactive hemagglutination (HA) site, a WEE complex-reactive HA site, and a WEE virus-specific HA site were identified. Spatial arrangement of these epitopes was determined by a competitive binding ELISA. Four competition groups defining three distinct antigenic domains were identified. Antibodies directed against four E1 epitopes were capable of precipitating the E1/E2 heterodimer from infected cells or purified virus disrupted with nonionic detergents. These same antibodies precipitated only E1 in the presence of 0.1% SDS. That E1 conformation was important was shown by the inability of antibodies specific for seven of the epitopes to bind to virus denatured in 0.5% SDS. As determined by equilibrium gradient analysis of virus-antibody mixtures, four epitopes were found to be fully accessible on the mature virion, three epitopes were inaccessible, and one epitope was partially accessible to antibody binding. Antibodies specific for three epitopes were able to passively protect mice from WEE virus challenge.

Animals↗
PubMed1988

Complex of brain D-phosphoglycerate mutase and gamma enolase and its reactivation by D-glycerate 2,3-bisphosphate.

The dissociabilities of dimeric gamma enolase, alpha enolase, and phosphoglycerate mutase of brain origin were tested using fluorescein isothiocyanate attached covalently to these enzymes. The dissociation constant of dimeric gamma enolase is lower (Kd = 0.03 microM) than that of the alpha enolase (Kd = 3 microM), while dimeric mutase seems to be nondissociable in the concentration range 0.1-10 microM, at pH 7.3 in 50 mM imidazole buffer at 20 degrees C. Interaction of neuron-specific gamma enolase with D-phosphoglycerate mutase was detected with the same fluorescence-labeling technique as well as by a kinetic analysis. The determined dissociation constant of the enolase-mutase complex was found to be in the range 5-40 microM, independent of the technique used. A mixed type of inhibition in the binding of D-glycerate-2-P and mutase to the D-glycerate-2-P binding site on enolase was observed in the absence of D-glycerate-2,3-P2. However, the inhibition of the enolase activity by brain D-phosphoglycerate mutase in the D-glycerate-2-P----phosphoenolpyruvate transformation is almost fully reverted by D-glycerate-2,3-P2, probably via the proper coordination of the active centers in the ternary complex of enolase, D-phosphoglycerate mutase, and their common intermediate, D-glycerate-2-P. The mechanism of intermediate transfer by consecutive enzyme pairs in a nondivergent metabolite flux (around the transformation of D-glycerate-2-P) is examined and conclusions of the present experiments are compared with the results of an extended analysis performed earlier with a divergent metabolite flux (around the transformation of multiusage triosephosphates, D-glyceraldehyde-3-P, and dihydroxyacetone phosphate).

2,3-Diphosphoglycerate↗
PubMed1999

Probing interactions between HIV-1 reverse transcriptase and its DNA substrate with backbone-modified nucleotides.

BACKGROUND: To gain a molecular understanding of a biochemical process, the crystal structure of enzymes that catalyze the reactions involved is extremely helpful. Often the question arises whether conformations obtained in this way appropriately reflect the reactivity of enzymes, however. Rates that characterize transitions are therefore compulsory experiments for the elucidation of the reaction mechanism. Such experiments have been performed for the reverse transcriptase of the type 1 human immunodeficiency virus (HIV-1 RT). RESULTS: We have developed a methodology to monitor the interplay between HIV-1 RT and its DNA substrate. To probe the protein-DNA interactions, the sugar backbone of one nucleotide was modified by a substituent that influenced the efficiency of the chain elongation in a characteristic way. We found that strand elongation after incorporation of the modified nucleotide follows a discontinuous efficiency for the first four nucleotides. The reaction efficiencies could be correlated with the distance between the sugar substituent and the enzyme. The model was confirmed by kinetic experiments with HIV-1 RT mutants. CONCLUSIONS: Experiments with HIV-1 RT demonstrate that strand-elongation efficiency using a modified nucleotide correlates well with distances between the DNA substrate and the enzyme. The functional group at the modified nucleotides acts as an 'antenna' for steric interactions that changes the optimal transition state. Kinetic experiments in combination with backbone-modified nucleotides can therefore be used to gain structural information about reverse transcriptases and DNA polymerases.

Acetylation↗
PubMed2003

D-arginase of Arthrobacter sp. KUJ 8602: characterization and its identity with Zn(2+)-guanidinobutyrase.

D-Arginase activity was found in the cells of an isolate, Arthrobacter sp. KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized. Its molecular weight was estimated to be about 232,000 by gel filtration, and that of the subunit was approximately 40,000 by SDS-PAGE, suggesting that the enzyme is a homohexamer. The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine. The V(max)/K(m) values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 micro mol/min/mg/mM, respectively. Accordingly, the enzyme is regarded as a kind of guanidinobutyrase [EC 3.5.3.7]. The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively. The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors. The enzyme contained about 1 g-atom of firmly bound Zn(2+) per mol of subunit, and removal of the metal ions by incubation with 1,10-phenanthroline resulted in loss of activity. The inactivated enzyme was reactivated markedly by incubation with either Zn(2+) or Co(2+), and slightly by incubation with Mn(2+). The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (M(r): 37,933). The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn(2+)-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).

Animals↗
PubMed1996

Acetylcholinesterase inhibition and protection by dizocilpine (MK-801) enantiomers.

The optical isomers of the N-methyl-D-aspartate (NMDA) receptor ion-channel blocker dizocilpine (MK-801) were shown to interact with electric eel and rat brain acetylcholinesterase (AChE) in a mixed competitive-noncompetitive way. The (-) form, pharmacologically less active, was the most potent of the two isomers as an AChE inhibitor (Ki for electric eel and rat brain AChE being 6.2 and 17.9 microM, respectively, compared with 200 and 450 microM, respectively, of the (+) form). Both enantiomers premixed with AChE preparations, dose-dependently protected the enzyme from inactivation by diisopropylfluorophosphate (DFP). The maximal protective effects against 40 and 10 microM DFP were in the ranges 10.7-23.8 and 19.5-31.4% of control enzymic activity for the (+) and (-) forms of dizocilpine, respectively. The extent of the protective effect against DFP was increased up to 80.1% of control enzymic activity for (-)-dizocilpine and to 38.4% for (+)-dizocilpine by diluting the enzymic mixtures 1000 times after treatment with the organophosphate agent. The two enantiomers added to AChE 15 min after DFP, failed to reactivate the enzyme. Finally, it was shown that (+)- and (-)-dizocilpine dose-dependently and competitively decreased the DFP bimolecular reaction constant, K(i). We conclude that dizocilpine exerts a protective action towards AChE against irreversible DFP inhibition, but the molecular mechanism of such an action is at present unclear.

Animals↗
PubMed1991

Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates.

Although most studies of protein phosphorylation have focused on intracellular protein kinases, evidence for protein kinase activity on the surface of several types of cells has been described. Evidence was recently provided for the existence of ecto-protein kinase activity on the surface of human neutrophils. Evidence for three distinct ecto-protein kinase activities was detected, one that phosphorylates endogenous surface proteins, one that phosphorylates exogenous substrates in a cAMP-independent manner and is released in the presence of substrate, and a low level of activity of one that phosphorylates exogenous Kemptide in a cAMP-dependent manner. To begin to elucidate its role in neutrophil function, we have characterized several properties of the releasable ecto-protein kinase activity on human neutrophils. This enzyme activity was inhibited by impermeant stilbene disulfonic acids, which are known to alter neutrophil function, as well as by impermeant sulfhydryl reactive agents. Enzyme activity was detectable at physiologic concentrations of Mg2+, but was higher in the presence of Mn2+. Protein kinase activity was strongly inhibited by heparin, whereas trifluoperazine, cAMP, and cGMP had little effect on kinase activity. Protein kinase activity was selectively removed from the cell surface by incubation with the ecto-kinase substrates casein and phosvitin, but the enzyme was not released by phosphatidylinositol-specific phospholipase C. Repeated exposure of neutrophils to substrate depleted ecto-protein kinase activity from the cell surface, but activity was rapidly restored by incubation in buffer lacking substrate. The released protein kinase had a Km for ATP of approximately 0.5 microM and a pH maximum between 7.0 and 7.5. At least four ecto-protein kinase substrates were detected in serum; vitronectin was identified as one of these substrates by immunoprecipitation studies. Although the exact role of ecto-protein kinase activity in neutrophil function remains undefined, the identification of vitronectin as a serum substrate suggests that it interacts with a physiologically important substrate.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Inactivation of intracellular proteolysis and cathepsin B enzyme activity by dehydroascorbic acid and reactivation by dithiothreitol in perfused rat heart.

The selective inhibition of some subcomponents of intracellular protein degradation was characterized under exposure to the cyclic multiketone thiol oxidizing agents dehydroascorbic acid (DHA) and alloxan. Proteins of the isolated perfused rat heart were labeled in vitro with a 10-min infusion of [3H] leucine, and subsequent release of radiolabeled amino acid from cell proteins was measured. As determined previously, four subcomponents of total proteolysis can be distinguished; the first three subcomponents are reversibly inhibited by the thiol-reactive agent diamide: (a) The rapid turnover proteins comprise most of the [3H] leucine release from 20 min to 3 hr after labeling. (b) Following 3 hr of degradation, the lysosomal (insulin-responsive) subcomponent comprises 35-40% of [3H] leucine release. (c) A third nonlysosomal (adrenergic-responsive) subcomponent comprises 35%. (d) A fourth nonlysosomal subcomponent consisting of 25% of [3H] leucine release is uninhibited by diamide. Infusion of supraphysiologic DHA (1 mM) or alloxan (1.5 mM) promptly mimicked the proteolytic inhibitory action of diamide on the first three subcomponents, but did not inhibit the diamide-resistant subcomponent. Infusion of a physiologic extracellular DHA concentration of 5 microM caused little or no change in proteolysis. The proteolytic inhibitory action of DHA (1 mM) could be reversed by concurrent infusion of dithiothreitol (DTT, 1.5 mM) simultaneously with continued DHA. DHA (1 mM) caused direct inhibition of the purified sulfhydryl proteinase cathepsin B (EC 3.4.22.1), which was reversible by subsequent excess DTT (5 mM). Results indicate that a nontoxic endogenous multiketone thiol oxidant can reversibly inhibit some proteolytic processes in viable tissue; however, approximately 25% of the observed proteolysis is uninhibited. Reversible inactivation of sulfhydryl proteinases, including cathepsin B, is among the possible multiple mechanisms of this DHA action.

Alloxan↗
PubMed1977

Creatine kinase: stability, inactivation, reactivation.

I determined the in vitro biological half-lives or decay constants for creatine kinase isoenzymes at various temperatures. Values at 37 degrees C are consistent with values reported by others in vivo, which suggests that in vivo irreversible inactivation is primarily thermal. Reversible inactivation appears to be an oxidation-reduction phenomenon. Proteins and some inactivators (urate, catecholamines) retard irreversible inactivation and preserve isoenzyme integrity. Dilution and thiols promote reversal of inactivity. Mercaptoethanol is the preferred thiol, particularly for storage and reactivation of isoenzyme MB. MB is sensitive to light and to freeze-thawing. I recommend that specimens be cooled promptly after drawing, that mercaptoethanol (10 mmol/liter) be added, and that they be stored refrigerated. Avoid prolonged exposure to light and freezing. A model of inactivation is proposed, which is based on the assumed existence of four monomer types: active, denatured, oxidized, and insulated. The model is consistent with dilution and thiol reactivation, lag phase variations, and subtype heterogeneity.

Catecholamines↗
PubMed1996

Effect of tamoxifen feeding on metabolic activation of tamoxifen by the liver of the rhesus monkey: does liver accumulation of inhibitory metabolites protect from tamoxifen-dependent genotoxicity and cancer?

Tamoxifen induces hepatocellular carcinomas in rats and is converted by rat hepatic cytochrome P450 enzymes into reactive metabolites capable of forming adducts with nucleic acids, proteins and chromosomal aberrations. In rats tamoxifen has also been shown to induce liver cytochrome P450 enzymes, to stimulate its own metabolism leading to greater covalent binding and to induce a higher degree of unscheduled DNA synthesis. This suggests that, at least in the rat, a sensitive species, tamoxifen may contribute significantly to its genotoxic and carcinogenic potential, by assisting its own metabolic activation. We have now investigated the effect of feeding tamoxifen to male and female Rhesus monkeys. A marked induction of the hepatic cytochrome(s) P450 is found in the monkey but, in spite of this, the in vitro metabolism of 7-ethoxyresorufin by microsomes from treated animals is markedly inhibited and so is the dealkylation of two other 7-alkoxyresorufin substrates. Evidence is presented for the accumulation in the liver of monkeys treated with tamoxifen of a powerful inhibitor of drug metabolism, and the inhibitor is identified as a metabolite of tamoxifen, its N,N-didesmethyl derivative. The level of 32P-postlabelled DNA adducts was considerably higher in rats given tamoxifen than in similarly treated monkeys. Also, whereas rats responded to tamoxifen treatment with a marked increase in covalent binding to microsomal protein, in the monkeys, where accumulation of the inhibitory metabolite in the microsomal fraction was also seen, covalent binding was not greater with microsomes from treated animals than in the corresponding controls. N,N-Didesmethyl-tamoxifen, added in vitro to human and rat microsomes, reduced significantly the extent of covalent binding, suggesting that the accumulation of the metabolite observed in the liver of primates may discourage the cytochrome P450-dependent conversion of tamoxifen into reactive derivatives and in this way protect against the formation of adducts. This mechanism may also contribute to protecting the primate against tamoxifen- induced liver cancer.

Animals↗
PubMed1980

Phospholipid reactivation of the purified plasma membrane ATPase of yeast.

The plasma membrane ATPase of the yeast Schizosaccharomyces pombe solubilized by lysolecithin and purified by centrifugation through a sucrose gradient is essentially inactive. The phospholipid distribution in the sucrose gradient indicates that inactivation of the ATPase may result from the partial delipidation occurring during purification. Taking into account the 100,000 daltons of the ATPase polypeptide, it is concluded that 74 mol of phospholipids are bound per mol of purified ATP monomer. The ATPase so purified is reactivated simply by mixing the enzyme with preformed lipid micelles or vesicles. Lysolecithins reactivate the enzyme at concentrations around the critical micellar concentration. Gel exclusion chromatography indicates that the enzyme binds reversibly to the lysolecithin micelles. On the other hand, lecithins of varying chain length and unsaturation reactivate the enzyme to different extents and with different efficiencies. In addition, from binding studies, it is observed that each saturated lecithin combines equally well with the ATPase. Using other diacylphospholipids no specificity for the polar head group is observed. Moreover, cardiolipin microvesicles is shown to bind all the protein but not to restore the enzyme activity. From lipid-reactivation titration curves. Arrhenius plots, and physical data of the phospholipids, it is concluded that the major parameter which governs the optimal reactivation of ATPase is the ability of the phospholipids to form amphipathic structures (micelles or vesicles) of sufficient fluidity and hydrophobicity. From these results, a coherent description can be provided for the protein-lipid interactions occurring during solubilization, purification, and the lipid reactivation of the yeast plasma membrane ATPase.

Adenosine Triphosphatases↗